As shown in WO 00/63403, immortalized human embryonic retina cells expressing at least an adenovirus E1A protein can be suitably used for the production of recombinant proteins.
Recombinant proteins having N-linked glycosylation produced in cells that express adenovirus E1A have a specific glycosylation profile, for instance characterized by the presence of Lewis-X structures, as described in WO 03/038100.
Another characteristic of the proteins produced thus far in E1A expressing cells appeared a relatively low galactosylation and low sialylation of the N-linked glycans (WO 03/038100). For certain purposes, this may be an advantage, but for other purposes, higher levels of galactosylation and preferably also sialylation may be beneficial.
For instance, erythropoietin (EPO) that is produced in cells expressing E1A, has a pronounced number of Lewis-X structures and a relatively low percentage of galactosylation and sialylation in the N-linked glycans (WO 03/038100), resulting in molecules that are very suitable for treatment of ischemia/reperfusion injuries, but may be less suitable for the treatment of anemia. For the treatment of anemia, it has been established that a high degree of sialylation of EPO is beneficial to increase the half-life of the EPO in serum of treated subjects, and thereby the time when the substance is active in increasing the red blood cell count (Goldwasser et al., 1974).
Hence, for the treatment of ischemia/reperfusion injuries, the expression of EPO in E1A-expressing cells has the potential advantage of a preferred glycosylation pattern of the produced EPO for this use. However, for other uses of EPO, different glycosylation patterns may be beneficial.
For other proteins similar situations may exist, i.e. for certain uses the specific glycosylation pattern observed upon expression in E1A-expressing cells may be highly beneficial, while for other purposes a different glycosylation profile may be more suitable.
Over-expressing of a sialyltransferase in a cell to increase sialylation of recombinant proteins produced in that cell has been described for other cell types (e.g., Grabenhorst et al., 1995; Minch et al, 1995; Jenkins et al, 1998; Zhang et al, 1998; Weikert et al, 1999; Fukuta et al., 2000; Prati et al., 2000). It was however not known before whether this approach could also lead to desired results in E1A-expressing cells, given the complexities of glycosylation and the still unclarified role of E1A therein (WO 03/038100). In particular, the interplay and potential competition between the various glycosyltransferases and other actors in the glycosylation process in cells that express E1A, rendered the outcome of over-expression of a sialyltransferase in such cells unforeseen and unpredictable in terms of glycosylation patterns of proteins thus produced.
For the purpose of broadening the potential use spectrum of recombinant proteins produced in E1A-expressing cells, it could be beneficial to increase the galactosylation and sialylation of such proteins. It is an object of the present invention to provide methods to accomplish this. The invention further aims at providing novel erythropoietin compositions obtainable from E1A-expressing cells.
It is another object of the invention to provide methods to decrease the average content of LacdiNAc structures on proteins recombinantly expressed in a cell, for instance a cell expressing E1A of an adenovirus.